Polymer-protein conjugation, particularly conjugation with polyethylene glycol (also known as PEGylation), is a well-established mean for increasing the circulation time, reducing antigenicity and improving the stability of proteins. Conjugation thus often leads to improved therapeutic benefits of the proteins as compared to non-conjugated proteins. Conjugation of proteins with polyethylene glycol (PEG) was shown to be applicable for various clinical uses (Duncan et al. Biomacromol., 2008, 9, 1146-1154).
Conjugation of FGF2 with PEG was described in Saik et al. (Acta Biomateri., 2011, 7, 133-143). Using an injured spinal cord model, the local administration of an FGF2-PEG conjugate provided an improved tissue penetration of the FGF2 (Kang et al, J. Cont. Release, 2010, 144, 25-31). Wu et al. (Prot. Expr. and Puri., 2006, 48, 24-27 and J. Chrom. A, 2007, 1161, 51-55) described the conjugation of PEG with modified FGF2 in which 3 out of 4 cysteine residues were replaced by serine residues. In these studies, the PEG-modified FGF2 conjugate retained the heparin binding capabilities of the modified FGF2 and partly retained its mitogenic activity. The conjugate also gained heat stability. US patent application No. 2004/0136952 discloses a method of synthesizing conjugates of synthetic water-soluble polymers with certain bioactive components, which conjugates retain high receptor binding activity. Specifically conjugates of PEG with Interferon-alpha, Interleukin-2, EGF and TGF-1 were disclosed. Conjugation of modified FGF21 with PEG was disclosed in U.S. patent application No. 2008/0255045. FGF21 was modified by introducing substitutions with unnatural amino acids that enabled its conjugation with azido-PEG. The pharmacokinetic profile of conjugated FGF21 was significantly improved as compared to non-conjugated FGF21. Conjugation of FGF1 with G5-polyamidoamine dendrimer was described in Thomas et al. (Bioorg. Med. Chem. Lett., 2010, 20(2), 700-703). It was suggested that multivalent G5-FGF nanoparticles may serve as a platform for cytosolic as well as nuclear drug delivery into tumor cells, and as an FGF delivery agent for inducing angiogenesis during wound healing.
Despite the advantages of conjugating proteins with polymers such as PEG, repeated administration of such conjugates was shown to result in the accumulation of the administered conjugate, mainly due to non-biodegradability of the polymers. The accumulation may lead to some potential problems including vacuolization, lysosomal storage diseases and, at high concentration, may also induce other pathological metabolic changes (Ferguson, Inter. J. Pharamc., 2010, 402(1-2), 95-102).
Hyaluronic Acid (HA)
Hyaluronic acid (hyaluronan, HA) is a glycosaminoglycan found in the extracellular matrix of all connective tissues. HA is known to bind specifically proteins in the extracellular matrix and on the cell surface. The unique viscoelastic properties of HA combined with its biocompatibility, immunoneutrality and its biodegradability has led to its use in a variety of clinical applications such as eye surgery and visco-supplementation of joints.
HA was implicated for utility as a carrier of cells, or growth factors for applications of bone deficiencies. US patent application No. 2004/0176295 provides a bone growth composition comprising a mixture of HA and an FGF. US patent application No. 2008/0193425 discloses that when hyaluronic acid is administered in addition to FGF18, the effects on chondrocyte proliferation and production of matrix were found to be greater than administration of FGF18 or hyaluronic acid alone. Prestwich (Biomaterials from Chemically modified Hyaluronan, Glycoforum; http://www.glycoforum.gr.jp/science/hyaluronan/HA18/HA18E.html) discussed the conjugation of hyaluronic acid with drugs (e.g. Taxol®). US patent application No. 2011/0212901 provides a hydrophobic group-introduced into HA derivative and a pharmaceutical composition comprising same. Conjugation of HA with peptide agonists of Formyl peptide receptor-like 1 (FPRL1) for increasing their half life circulation and/or their bioavailability was disclosed in Oh et al. (Bioconjugate Chem., 2008, 19, 2401-2408). In another study, HA was conjugated to anti-flt1 peptide (Oh et al, Biokmaterials, 2009, 20, 6026-6034). J-Hyun Kong et al. (Biomaterials, 2010, 31, 4121-4128) described the conjugation of HA with exendin 4 (Byetta®), a 39 amino acid peptide which was exploited for the treatment of type 2 diabetes. In this study, the in vitro serum stability of exendin 4 was improved by a factor of 20 while maintaining its biological activities. U.S. Pat. No. 7,034,127 disclosed methods of conjugating biologically active substances, particularly α-interferon, with HA. The HA-interferon conjugate was biologically active. Further disclosed in U.S. Pat. No. 7,034,127 is the preparation of HA conjugates with epidermal growth factor (EGF), anti-BSA antibody, cytochrome C and avidin. Liu et al. (J Biomed Mater. Res., 2002, 62(1): 128-35) demonstrated hyaluronate-heparin conjugate gels for the delivery of FGF-2. U.S. Pat. No. 6,288,043 to Liu et al. disclosed an injectable composition for promoting bone and/or cartilage growth comprising hyaluronate-heparin conjugates. These conjugates have inherent binding sites for members of the FGF family. Ferguson, (Inter. J. Pharm., 2010, 402(1-2), 95-102) demonstrated the conjugation of HA with EGF with a very low yield (12%). The conjugate was inactive, even after treatment with hyaluronidase which degrades HA. In the same study, HA-trypsin conjugate retained its activity. WO 2001/05434 disclosed the conjugation of HA with Interleukin-1 (II-1) receptor antagonist, osteoprotegrin and leptin. Several protein conjugates showed improved efficacy, longer circulation time, higher water solubility and reduction in adverse injection site reactions. Interlukin-1β and tumor necrosis factor monoclonal antibodies were covalently modified with HA and carboxy methyl cellulose (CMC). These conjugates were capable of binding pro-inflammatory cytokines (Sun et al, Mol. Pharma., 2010, 7, 1769-1777). EP 1790665 disclosed a process for producing water-soluble HA modification. Further disclosed is a conjugate of the modified HA with a drug, a protein, a peptide, a nucleic acid, or a low-molecular-weight compound including IgG Fab and GLP-1. WO 2008/081463 to some of the inventors of the present application discloses the water-soluble reactive esters of carboxy polysaccharides and derivatives thereof and the formation of water-soluble covalent fibrinogen conjugates, including HA-fibrinogen conjugates. In an effort to obtain long-acting formulation of biopharmaceuticals, US patent application No. 2010/0210509 discloses a long acting conjugate of peptide with HA derivative having a long-term stability and a high efficacy, and a method of preparing said conjugate.
Fibroblast Growth Factors
Fibroblast growth factors (FGFs) comprise a large family of evolutionarily conserved polypeptides involved in a variety of biological processes including morphogenesis, angiogenesis, and tissue remodeling as well as in the pathogenesis of numerous diseases. The various members of this family stimulate the proliferation of a wide spectrum of cells, including those deriving from mesenchymal, endothelial, epithelial and neuroectodermal origin. FGFs are expressed in a strict temporal and spatial pattern during development and have important roles in patterning and limb formation (Ornitz, Bioassays, 2000, 22, 108-112). All members of the FGF family share a homology core domain of about 120 amino acids, 28 aa residues are highly conserved and four are identical. The adjacent N- and C-termini are of variable length and share limited homology. The core domain comprises both the primary receptor binding sites and a heparin-binding domain, which are distinct from each other (Ornitz et al, Gen. Biol., 2001, 2(3), 3005.1-3005.12). Because of their wide ranging and potent activities, FGFs are pursued as therapeutic agents for a number of different indications, including wound healing, bone fractures, skin conditions, tissue protection, repair, and the induction of angiogenesis during myocardial infarction and ischemia, inflammatory conditions, neurological conditions, and diabetes. WO 2012/038953 to some of the inventors of the present application discloses FGF-18 variants having increased receptor specificity for cartilage repair and treatment of degenerative joint disease.
FGFs were previously conjugated or incorporated into drug delivery systems. US patent application No. 2008/0176790 discloses conjugates of modified FGF peptides, particularly FGF20 and FGF21 peptides with PEG polymer. The FGF peptides are modified with N-linked or O-linked glycosylation site(s). US patent application No. 2011/0015345 discloses modified FGF-23 polypeptides linked to a water-soluble polymer, particularly PEG.
There remains an unmet need for FGFs with sustained release or prolonged effects for use for example in wound healing, osteoarthritis or bone repair conjugates. These attributes are provided by conjugates of FGFs with carboxy polysaccharides, particularly HA, which provide improved bioactivity and stability of the FGFs for various therapeutic applications.